(A) Effects of PKA on the complex formation of Axin, β-catenin, GSK-3β, and APC. After L cells were incubated with Bt₂cAMP and IBMX for 2 h, the lysates were probed with the indicated antibodies. The lysates were immunoprecipitated with the anti-Axin antibody, and the immunoprecipitates were probed with the indicated antibodies. IP, immunoprecipitation. (B) Effects of PKA on the stability of Axin. L cells were treated with Bt₂cAMP and IBMX or Wnt-3a for the periods indicated. The lysates were probed with the indicated antibodies.

Activation of Tcf-4 by PKA. (A) Activation of Tcf-4 by PGE₁ and Bt₂cAMP. L cells transfected with pEF-BOS-HA/hTcf-4E (0.1 μg) and TOP-fos-Luc (0.5 μg) or FOP-fos-Luc (0.5 μg) were treated with PGE₁, Bt₂cAMP and IBMX, or Wnt-3a for 8 h. The luciferase activity was measured and expressed as the fold increase compared with the level observed in cells without treatment. (B) Effects of PKA on the Tcf-4 activity toward a natural Tcf-responsive promoter. pEF-BOS-HA/hTcf-4E (0.1 μg) or pEF-BOS-HA/hTcf-4E (Δ1-53) (0.1 μg) was transfected into L cells with Axin2-Luc (0.5 μg). The cells were treated with PGE₁, Bt₂cAMP and IBMX, or Wnt-3a in the presence or absence of H-89 for 8 h. The luciferase activity was measured and expressed as the fold increase compared with the level observed in the cells without treatment. (C) Effects of PKA on expression of the Axin2 mRNA. Total RNA from HEK-293 cells stimulated with Bt₂cAMP and IBMX or Wnt-3a for 2 h in the presence or absence of H-89 was subjected to quantitative RT-PCR. The results shown were normalized by the GAPDH mRNA levels, and the corresponding quantification results for the Axin2 mRNA levels were expressed as the fold increase compared with that of cells without treatment. The results shown are means ± standard errors from four independent experiments.

Inhibition of degradation of β-catenin by PKA. (A) Effects of PKA on expression of the β-catenin mRNA. HEK-293 cells were treated with Bt₂cAMP and IBMX or Wnt-3a for 2 h and then subjected to quantitative RT-PCR. The results shown were normalized using the GAPDH mRNA levels, and the corresponding quantification results for cyclin D1 and β-catenin mRNA levels were expressed as the fold increase compared with those in the absence of Bt₂cAMP and Wnt-3a. The results shown are means ± standard errors from four independent experiments. (B) Effects of PKA on protein degradation of β-catenin. L cells were treated with Bt₂cAMP and IBMX or Wnt-3a for the periods indicated in the presence of 25 μg/ml cycloheximide. The β-catenin protein levels were normalized using the GSK-3β protein level and expressed as arbitrary units. (C) Effects of PKA on the protein levels of IκBα. HEK-293 cells were preincubated with or without Bt₂cAMP and IBMX for 1 h and then stimulated with 10 ng/ml TNF-α for 10 min. The cytosol was probed with the indicated antibodies. The results shown are representative of three independent experiments.

Phosphorylation of GSK-3β and β-catenin by PKA. (A) Regulation of GSK-3β activity by PKA in the Axin complex. After L cells stably expressing Myc-rAxin were treated with Bt₂cAMP and IBMX for the periods indicated, the activities of total GSK-3β (•) and GSK-3β complexed with Axin (○) were measured. The results shown are expressed as a percentage of the activity of GSK-3β at time zero and are means ± standard errors from three independent experiments. (B) Phosphorylation of β-catenin by PKA, CKIα, and GSK-3β in vitro. His₆-β-catenin (2 μg of protein) was incubated with or without 0.1 μM GST-GSK-3β in the presence or absence of MBP-rAxin (400 ng of protein) for 30 min after prephosphorylation by 0.125 μM MBP-CKIα or 20 U of PKAc. The top panel is an autoradiograph that shows the total phosphate incorporated into His₆-β-catenin. The phosphorylation states of His₆-β-catenin were detected with the indicated antibodies. (C) Phosphorylation of β-catenin by PKA in intact cells. HEK-293 cells were stimulated with Bt₂cAMP and IBMX or Wnt-3a for the periods indicated. The cytosol was probed with the indicated antibodies. The results shown are representative of three independent experiments.

Accumulation of β-catenin induced by PKA activation in intact cells. (A) After L cells were preincubated with or without 10 μM H-89 for 30 min, the cells were stimulated with 50 ng/ml PGE₁ or Wnt-3a-conditioned medium for 2 h. The lysates were probed with the indicated antibodies. Ab, antibody. (B) Wild-type L cells (L/WT) and L cells stably expressing β₂AR (L/β₂AR) were treated with 10 μM isoproterenol in the presence or absence of H-89 for 2 h. The lysates were probed with the indicated antibodies. (C) HEK-293 cells were stimulated with 1 mM Bt₂cAMP and/or 0.1 mM IBMX for 2 h (lanes 1 to 4). Before HEK-293 cells were stimulated with the indicated reagents, the cells were preincubated with or without 10 μM H-89 for 30 min (lanes 5 to 10). The cytosol was probed with the indicated antibodies. (D) RNA interference for PKAc. L cells were transfected with dsRNA oligonucleotides for PKAcα and PKAcβ. The cells were stimulated with Bt₂cAMP and IBMX for 2 h, and the lysates were probed with the indicated antibodies. (E) The cytoplasm and nuclear fractions from L cells stimulated with Bt₂cAMP and IBMX or Wnt-3a for 2 h were probed with the indicated antibodies. (F) HEK-293 or L cells were stimulated with Bt₂cAMP and IBMX, Wnt-3a, or both for 2 h. The cytosol of HEK-293 cells or the lysate of L cells was probed with the indicated antibodies. The results shown are representative of three independent experiments.

Identification of phosphorylation site of β-catenin that impairs ubiquitination. (A) Phosphorylation site of β-catenin. Two possible phosphorylation sites of β-catenin by PKA are indicated. Wild-type His₆-β-catenin, His₆-β-catenin^S675A, and His₆-β-catenin^T510A (2 μg) were phosphorylated by PKAc, and the samples were subjected to autoradiography (lanes 1 to 3). Wild-type His₆-β-catenin and His₆-β-catenin^S675A were incubated with or without PKAc. The samples (10 ng) were probed with the anti-β-catenin and anti-pSer675 antibodies (lanes 4 to 7). L cells were treated with Bt₂cAMP, PGE₁, or Wnt-3a. The lysates were immunoprecipitated with the anti-β-catenin antibody, and the equivalent amounts of β-catenin were probed with the anti-pSer675 antibody (lanes 8 to 11). After HEK-293T cells expressing HA-β-catenin (lanes 12 and 13) or HA-β-catenin^S675A (lanes 14 and 15) were treated with Bt₂cAMP, the lysates were immunoprecipitated with the anti-HA antibody and the equivalent amounts of β-catenin were probed with the indicated antibodies. (B) Pulse-chase analysis of β-catenin. COS cells expressing wild-type HA-β-catenin (circles), HA-β-catenin^S675A (squares), or HA-β-catenin^T510A (triangles) were subjected to pulse-chase analyses in the presence (filled symbols) or absence (open symbols) of Bt₂cAMP and IBMX. β-Catenin and its mutants were immunoprecipitated with the anti-ΗΑ antibody, and the incorporation of ³⁵S into β-catenin was scanned using the Molecular Dynamics Storm imaging system. Signals were quantified using ImageQuant software (Molecular Dynamics) and expressed as the percentage of the value of time zero. The results shown are means ± standard errors of five independent experiments. (C) Effects of PKA on ubiquitination of β-catenin^S675A. The assay conditions were the same as those for Fig. 6F except that β-catenin^S675A was used. (D) Effects of PKA on the Tcf-4 activity through β-catenin^S675A. pCGN/β-catenin (0.5 μg), pCGN/β-catenin^T510A (0.5 μg), pCGN/β-catenin^S675A (0.5 μg), or pCGN/β-catenin^SA (0.5 μg) was transfected into L cells with Axin2-Luc (0.5 μg) and pEF-BOS-HA/hTcf-4E (0.1 μg) or pEF-BOS-HA/hTcf-4E (Δ1-53) (0.1 μg) in the presence or absence of pCGN/PKAc (0.1 μg). After 36 h, the luciferase activity was measured and expressed as the fold increase compared with the level observed in the cells transfected with Axin2-Luc and pEF-BOS-HA/hTcf-4E. The results shown are means ± standard errors from four independent experiments. WT, wild type.

Inhibition of ubiquitination of β-catenin by PKA. (A) Decrease in ubiquitination of β-catenin by PKA in intact cells. HEK-293 cells expressing HA-ubiquitin and FLAG-Fbw1 were incubated with (lanes 3, 6, and 9) or without (lanes 1, 2, 4, 5, 7, and 8) Bt₂cAMP and IBMX for 2 h in the presence of 10 μM lactacystin. The lysates were probed with the anti-HA antibody (lanes 1 to 3). After the lysates had been immunoprecipitated with the anti-Myc (lanes 4 and 7) or anti-β-catenin (lanes 5, 6, 8, and 9) antibody, the immunoprecipitates were probed with the anti-HA (lanes 4 to 6) or β-catenin (lanes 7 to 9) antibody. The arrowhead indicates the position of β-catenin. (B) Complex formation of Fbw1 and β-catenin. L cells expressing FLAG-Fbw1 were stimulated with Bt₂cAMP and IBMX for 2 h. The lysates were immunoprecipitated with the anti-FLAG antibody. The immunoprecipitates were probed with the indicated antibodies. (C) Effects of Fbw1 on PKA-dependent nuclear localization of β-catenin. HEK-293 cells expressing FLAG-Fbw1 and HA-β-catenin were treated with Bt₂cAMP and IBMX for 2 h. The nuclear fractions were prepared and probed with the indicated antibodies. (D) Effects of Fbw1 on PKA-dependent Tcf-activity. pEF-BOS-HA/hTcf-4E (0.1 μg) and Axin2-Luc (0.5 μg) were transfected into L cells with pEF-BOS-FLAG/Fbw1 (0.1 μg). The cells were treated with Bt₂cAMP and IBMX, or Wnt-3a, for 8 h. The luciferase activity was measured and expressed as the fold increase compared with the level observed in the cells without treatment. The results shown are means ± standard errors from four independent experiments. (E) The recombinant SCF^Skp1 complex was purified from Sf9 cell lysate expressing His₆-T7-Skp1, HA-Cul1, GST-Myc-Fbw1, and Myc-Rbx1. The proteins were detected with their antibodies. (F) Decrease in ubiquitination of β-catenin by PKA in vitro. Purified His₆-β-catenin was incubated with MBP-rAxin, His₆-CΚIɛ, and GST-GSK-3β in the presence (lanes 1 and 2) or absence (lane 3) of ATP in the kinase reaction mixture for 2 h at 30°C. The samples were further subjected to the ubiquitination assay in the presence (lanes 1 and 3) or absence (lane 2) of His₆-Myc-ubiquitin for 1 h at 30°C. Before the ubiquitination assay, His₆-β-catenin was preincubated with or without PKAc in the presence or absence of H-89 (lanes 4 to 6). (G) Effects of pretreatment of the SCF complex with PKA on ubiquitination of β-catenin. The assay conditions were the same as those in panel F, except that recombinant SCF^Skp1 complex pretreated with PKAc for 1 h at 30°C was used. The results shown are representative of three independent experiments.